Novel Multiphoton Imaging Techniques Reveal Brain’s Secrets

Two-photon microscopy has surpassed confocal imaging for the study of intact, living tissue. Three-photon imaging is not far behind and is already allowing researchers to image at depths.

FAROOQ AHMED, CONTRIBUTING EDITOR

Nearly two decades ago, Winfried Denk and Karel Svoboda argued in the journal Neuron that “multiphoton imaging is more than a gimmick”1. At the time, confocal microscopy — combined with advancements in fluorescent reporter systems and an increase in desktop computing power — had set a high standard for 3D imaging of biological specimens. But Denk, a German physicist at Bell Laboratories in Murray Hill, N.J., who developed two-photon imaging in 1990 when he was a

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Published: January 2019

Glossary

excitation: 1. The process by which an atom acquires energy sufficient to raise it to a quantum state higher than its ground state. 2. More specifically with respect to lasers, the process by which the material in the laser cavity is stimulated by light or other means, so that atoms are converted to a semistable state, initiating the lasing process.
photobleaching: Photobleaching is a phenomenon in which the fluorescence of a fluorophore (a fluorescent molecule or dye) is permanently reduced or eliminated upon prolonged exposure to light. This process occurs due to the photochemical destruction or alteration of the fluorophore molecules, rendering them non-fluorescent. Key points about photobleaching: Mechanism: Photobleaching is typically a result of chemical reactions induced by the absorbed photons. The excessive light exposure causes the...